Oscillations of cytosolic Ca²⁺ ([Ca²⁺]_c) are widely observed in response to extracellular signals that generate inositol 1,4,5-trisphospate (InsP₃). One of the first demonstrations of this phenomenon was by Cobbold and coworkers (Woods et al. 1986). The mechanism underlying the generation these [Ca²⁺]_c oscillations has been the subject of much discussion (Thomas et al. 1996; Berridge et al. 1999). The predominant view is that [Ca²⁺]_c oscillations arise from positive and negative feedback effects of Ca²⁺ and InsP₃ on the inositol 1,4,5-trisphosphate receptor (InsP₃R). Thus the InsP₃R acts as a Ca²⁺-induced Ca²⁺ release channel, giving rise to regenerative cycles of Ca²⁺ release and reuptake at a fixed elevated level of intracellular InsP₃. While there is clear evidence that [Ca²⁺]_c oscillations can be generated in such a manner at the level of the InsP₃R (Meyer & Stryer, 1988; Hirose et al. 1999), it appears unlikely that this can account for all of the properties of InsP₃-dependent [Ca²⁺]_c oscillations observed in intact, agonist-stimulated cells. For example, [Ca²⁺]_c oscillations elicited by vasopressin and α-adrenergic agonists in the liver appear as baseline-separated spikes of constant frequency, with interspike periods that can last for many minutes. One way in which the properties of [Ca²⁺]_c oscillations might be enriched, is through the interplay of multiple interacting oscillators. Of particular interest in this respect is the possibility that Ca²⁺ may have feedback effects on InsP₃ metabolism, to cause oscillations in the level of InsP₃ during agonist stimulation (Meyer & Stryer, 1988). Studies with a Plextrin Homolgy Domain-Green Fluorescent Protein construct have provided evidence for such oscillations of phospholipase activity and cytosolic InsP₃. It has also been suggested that other intracellular organelles or plasma membrane Ca²⁺ transport pathways may participate in the generation of [Ca²⁺]_c oscillations. In this context, mitochondria are of particular interest.

In this presentation, we will discuss some new evidence for a role of [Ca²⁺]_c feedback on InsP₃ metabolism in the genesis of [Ca²⁺]_c oscillations, and will revisit the role of mitochondria. In order to investigate the potential role of InsP₃ oscillations, we have designed a cytosolic InsP₃ buffer based on the ligand binding domain of the rat type I InsP₃ receptor (LBD), to buffer InsP₃ in the physiological range. This InsP₃ buffer is composed of green fluorescent protein fused in-frame to the N-terminal ligand-binding domain of the rat type I InsP₃ receptor (GFP-LBD). We demonstrate that this construct, when transiently expressed in a variety of cells, is able to suppress [Ca²⁺]_c oscillations without preventing the InsP₃-dependent elevation of [Ca²⁺]_c. Expression of a mutated (R265Q) GFP-LBD, which is unable to bind InsP₃, had no effect on [Ca²⁺]_c oscillations. Taken together with direct measurements of InsP₃ in these cells, the data demonstrate that GFP-LBD functions as an InsP₃ buffer, and that InsP₃ oscillations are not only a consequence of [Ca²⁺]_c oscillations, but actually play a causal role in generating [Ca²⁺]_c oscillations.

Mitochondria generally maintain a relatively low level of matrix Ca²⁺ ([Ca²⁺]_m), and are not considered to be a primary Ca²⁺ source for the generation of [Ca²⁺]_c signals. Nevertheless, there is much functional evidence to suggest that mitochondria are often closely associated with other Ca²⁺ mobilization pathways, including InsP₃Rs. Changes in [Ca²⁺]_m often parallel the spatiotemporal organization of cytosolic Ca²⁺ signals. Mitochondrial Ca²⁺ uptake has important effects on mitochondrial metabolism, which serve to co-ordinate ATP production with the activation of Ca²⁺-dependent processes occurring in the cytoplasm. However, mitochondrial Ca²⁺ uptake can also modify the bulk flow of Ca²⁺ into the cytosol and alter the Ca²⁺ feedback effects that regulate Ca²⁺ channels in both the plasma membrane and intracellular Ca²⁺ storage compartments. Based on the properties of mitochondrial Ca²⁺ uptake and release pathways, the interactions with other Ca²⁺ mobilization pathways are believed to reflect a strategic localization of the mitochondria close to the primary Ca²⁺ channels. This apparently facilitates efficient Ca²⁺ translocation into the mitochondrial matrix, despite the relatively low [Ca²⁺]_c affinity of the mitochondrial uniporter. Mitochondrial Ca²⁺ handling modifies the regenerative activation of InsP₃R Ca²⁺ channels by Ca²⁺, and appears to play an important role in modulating the spatial and temporal properties of [Ca²⁺]_c oscillations.

This work was supported by funding from NIH.

All procedures accord with current National guidelines.